The directed synthesis of biological macromolecules is one of the great achievements of biochemistry. With the advent of recombinant DNA (rDNA) technology, it has become possible to harness the catalytic machinery of the cell to produce a desired protein. This can be achieved within the cellular environment or in vitro using extracts derived from cells.
Cell-free protein synthesis offers several advantages over conventional, in vivo, protein expression methods. Cell-free systems can direct most, if not all, of the metabolic resources of the cell towards the exclusive production of one protein. Moreover, the lack of a cell wall and membrane components in vitro is advantageous since it allows for control of the synthesis environment. For example, tRNA levels can be changed to reflect the codon usage of genes being expressed. The redox potential, pH, or ionic strength can also be altered with greater flexibility than in vivo since we are not concerned about cell growth or viability. Furthermore, direct recovery of purified, properly folded protein products can be easily achieved.
In vitro translation is also recognized for its ability to incorporate unnatural and isotope-labeled amino acids as well as its capability to produce proteins that are unstable, insoluble, or cytotoxic in vivo. In addition, cell-free protein synthesis may play a role in revolutionizing protein engineering and proteomic screening technologies. The cell-free method bypasses the laborious processes required for cloning and transforming cells for the expression of new gene products in vivo, and is becoming a platform technology for this field.
Despite all of the promising features of cell-free protein synthesis, its practical use and large-scale implementation has been limited by several obstacles. Paramount among these are short reaction times and low protein production rates, which lead to poor yields of protein synthesis and excessive reagent cost. Additionally, expensive reagents are required, and conventional methods are inefficient in the use of these expensive reagents.
Particularly useful reactions combine in vitro transcription and translation, thereby providing a direct link between a DNA coding sequence and the protein product. However, the additional requirement for reagents to produce mRNA add to the overall cost of the reaction. Recent publications have discussed many different strategies for cost reduction of in vitro transcription reactions, including reusing DNA templates and employing fed batch protocols. For example, see Kern and Davis (1997) “Application of Solution Equilibrium Analysis to in-Vitro RNA Transcription” Biotechnology Progress 13:747-756; Kern and Davis (1999) “Application of a Fed-Batch System to Produce RNA by In-Vitro Transcription” Biotechnology Progress 15:174-184.
Improvements are required to optimize in vitro transcription/translation systems. The continuous removal of the inhibitory by-product(s) as well as the continuous supply of substrates for synthesis may enable continuous or semicontinuous reaction systems to support synthesis over long reaction periods. However, these approaches may also result in inefficient use of substrates and therefore in high costs. Elucidation of inhibitory products, and prevention of their synthesis is of great interest for development of in vitro synthetic systems. Also important is the reduction of reagent costs. With present technology, the major reagent costs include the source of chemical energy, enzymes, DNA template, and NTPs. Methods of decreasing these costs while enhancing yield are of great interest.
Relevant Literature
U.S. Pat. No. 6,337,191 B1, Swartz et al. Kim and Swartz (2000) Biotechnol Prog. 16:385-390; Kim and Swartz (2000) Biotechnol Lett. 22:1537-1542; Kim and Choi (2000) J Biotechnol. 84:27-32; Kim et al. (1996) Eur J Biochem. 239: 881-886; Kim and Swartz (2001) Biotechnol Bioeng 74:309-316; Davanloo et al., Proc Nat'l Acad Sci USA 81:2035-2039 (1984); Datsenko et al., Proc Nat'l Acad Sci USA 97:6640-6645 (2000); Jewett et al. (2002) Prokaryotic systems for in vitro expression, in Gene Cloning and Expression Technologies (Weiner, M. P. and Lu, Q.: eds.), Eaton Publishing, Westborough, Mass., pp. 391-411; Spirin et al., Science 242:1162-1164 (1988).
Cunningham and Ofengand (1990) Biotechniques 9:713-714 suggest that adding inorganic pyrophosphatase results in larger reaction yields by hydrolyzing the pyrophosphate that accumulates. Pyrophosphate is inhibitory because the pyrophosphate complexes with the free magnesium ions leaving less available for the transcription reaction.
Breckenridge and Davis (2000) Biotechnology Bioengineering: 69:679-687 suggest that RNA can be produced by transcription from DNA templates immobilized on solid supports such as agarose beads, with yields comparable to traditional solution-phase transcription. The advantage of immobilized DNA is that the templates can be recovered from the reaction and reused in multiple rounds, eliminating unnecessary disposal and significantly reducing the cost of the DNA template.
U.S. Pat. No. 6,337,191 describes in vitro protein synthesis using glycolytic intermediates as an energy source; and U.S. Pat. No. 6,168,931 describes enhanced in vitro synthesis of biological macromolecules using a novel ATP regeneration system.